Increasingly, histological examination of biological specimens is being performed under automated conditions. Equipment and protocols have been developed for many automated routine stains and there is increased interest in automating non-routine, complex or special stains.
During manual staining, staining reagents can be safely prepared and stored according to conventional laboratory procedures. Corrosive reagents required in some staining protocols, for example, are stored according to safety guidelines, often in glass containers, and are dispensed as needed during the staining procedure.
In automated staining systems, however, reagent packs, valve parts, and other instrument components often are constructed from plastic materials, for example from clear and flexible elastomeric polymers. Such materials are incompatible with some of the chemical reagents required in staining protocols and the problem is exacerbated when the staining reagent is stored at room temperature and/or for prolonged periods of time. Storing staining reagents in liquid dispensers fabricated from plastic materials with which they are incompatible can result in leaks, loss of liquid, loss of accurate dispensing, as well as ii safety hazards.
Therefore, a need exists for automated staining procedures which eliminate or minimize the aforementioned problems.
The methods of the present invention are directed to staining processes. In particular, the methods described herein are related to combining precursors of a staining reagent and contacting the combined precursors with a biological specimen. The methods of the invention can be carried out in an automated staining process which uses liquid dispensers to dispense staining reagents. In one embodiment of the invention, the staining protocol requires the use of a staining reagent which is incompatible with at least one material, (e.g., plastic) employed in the fabrication of the liquid dispenser. One example of a staining reagent is chromic acid which is used in Grocott""s Modification of Gomori""s Methenamine Silver (GMS) stain which is typically employed to detect fungi in cytological or histological specimens.
In one embodiment, the method of the invention includes providing a biological specimen, for instance on a microscope slide, and dispensing onto the biological specimen two or more precursors of the staining reagent. The precursors are delivered from separate liquid dispensers. When combined, such as, for example by contacting one another on the biological specimen, the precursors either form the staining reagent in situ or have the same effect on the biological specimen as that of the staining reagent. Preferably, the precursors are more compatible with the liquid dispensers than is the staining reagent. In the case of chromic acid, an oxidizing precursor, such as, for instance, a source of chromate ions, and a source of hydrogen ions can be dispensed separately onto the biological specimen. The chromate ions and hydrogen ions form in situ chromic acid or, alternatively, have the same effect on the biological specimen as that of chromic acid, and the stain process proceeds according to the standard protocol.
In alternative embodiments, precursors of chromic acid also can be employed in staining procedures-which are carried out manually.
The invention has many advantages. For example, special stains requiring strong oxidizing agents or other corrosive staining reagents can be automated without changing the design or materials employed to fabricate the liquid dispensers. The precursors employed can be pre-packed and stored for longer periods or at higher temperatures than previously possible with the staining reagent itself. Safety hazards, leaks and inconsistent delivery of reagent amounts are minimized.